Pancreatic Trypsin Activates Human Promatrix Metalloproteinase-2

• Published in: Journal of molecular biology 2005-07, Vol.350 (4), p.682-698
• Main Authors: Lindstad, Rune I., Sylte, Ingebrigt, Mikalsen, Svein-Ole, Seglen, Per O., Berg, Eli, Winberg, Jan-Olof
• Format: Article
• Language: English
• Subjects: Alkylating Agents - pharmacology; Calcium - metabolism; Detergents - pharmacology; Enzyme Activation - drug effects; Enzyme Activation - physiology; Enzyme Precursors - metabolism; gelatinaseA; Humans; Isoelectric Point; Isoenzymes; Kinetics; Matrix Metalloproteinase 2 - metabolism; MT-MMP; Pancreas - enzymology; Pancreas - metabolism; Polyethylene Glycols - pharmacology; promatrix metalloproteinase-2; Protein Structure, Tertiary; TIMP; Tissue Inhibitor of Metalloproteinase-1 - metabolism; Titrimetry; Tosylphenylalanyl Chloromethyl Ketone - pharmacology; trypsin; Trypsin - metabolism
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2005.05.018

In contrast to the prevalent view in the literature hitherto, the present study shows that pancreatic trypsin can activate human promatrix metalloproteinase-2 (proMMP-2). It is shown that trypsin's ability to activate proMMP-2 is dependent on various environmental factors such as the level of exogenously added Ca 2+ and Brij-35, temperature, as well as trypsin concentration. The activation occurred as a sequential processing of the proenzyme, initially generating an active 62 kDa species. This was followed by successive truncation of the C-terminal domain, giving rise to active species of 56 kDa, 52 kDa and 50 kDa. Tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) prevented the trypsin-mediated C-terminal truncation, without affecting the generation of the 62 kDa species, while the presence of EDTA increased the rate of the trypsin-mediated activation of proMMP-2. MALDI-TOF MS analysis of the 50 kDa form indicated that trypsin generated active forms with either Lys87 or Trp90 as the N-terminal residue and Arg538 as a C-terminal residue. The trypsin-activated MMP-2 was active in solution against both synthetic and physiologic substrates, and the steady-state kinetic coefficients k cat, K m and k cat/ K m were determined for the enzyme activated either by APMA, membrane-type 1 matrix metalloproteinase (MT1-MMP) or trypsin. The trypsin-activated MMP-2 exhibited slightly lower k cat and k cat/ K m values as well as a slightly higher K i value against TIMP-1 compared to the enzyme activated by APMA or MT1-MMP. Docking studies of TIMP-1 revealed that the slightly weaker binding of the inhibitor to the trypsin-activated MMP-2 could be attributed to its shorter N terminus (Lys87/Trp90 versus Tyr81), as Phe83 and Arg86 interacted directly with the inhibitor. Our results suggest that the trypsin-activated MMP-2 possesses the catalytic and regulatory potential to be of significance in vivo.